Project Summary: Maintaining genome and epigenome stability is required in both mitotic and post-mitotic cells to prevent deleterious mutations that can cause diseases like cancer and neurodegenerative disorders. In proliferative cells, DNA replication needs to be coordinated with transcription and DNA repair to prevent the occurrence and inheritance of mutations. In terminally differentiated, non-replicating cells like neurons, chromatin has to be stable for long periods of time to maintain established transcriptional programs. With this proposal, we seek to understand the contributions of histone H3 variants to genomic and epigenomic stability during DNA replication and aging. Levels of replication- dependent H3.1/H3.2 variants and replication-independent H3.3 variants change drastically during DNA replication in proliferating cells and through time in post-mitotic cells. We have shown previously that H3.1 and H3.3 variants can have different biochemical functions on the epigenome in plants, which are mediated by a variable amino acid residue (position 31) in the N-terminal tail of these two highly conserved proteins. Through the study of the H3K27 methyltransferases ATXR5 and ATXR6 (ATXR5/6), we have demonstrated that newly synthesized H3.1 variants are specifically methylated by ATXR/6 during DNA replication, and that H3.1 variants unmethylated at K27 (H3.1K27me0), but not H3.3K27me0, induce genomic instability. We will investigate the biochemical and cellular mechanisms by which H3.1 regulates genomic stability in plants using genetic and biochemical screens for chromatin-interacting proteins that can discriminate between H3.1 and H3.3. We will also use a histone replacement strategy to analyze the roles of all histones in maintaining or disrupting genomic stability during DNA replication. Finally, we will replicate these experiments to study the role of H3 variants in maintaining heterochromatin in aging neurons using Drosophila as a model system. The high levels of similarity between H3.1, H3.3 and chromatin-modifying proteins across all eukaryotes suggest that the molecular mechanisms that will be uncovered through this work will be conserved in humans and involved in diseases caused by genomic or epigenomic instability.